Flow chamber and analyte detection method

ABSTRACT

A flow chamber and method for detecting the presence of one more cell produced analytes under flow conditions. The flow chamber includes two compartments separated by a permeable membrane on which a plurality of cells may be positioned. The permeable membrane shields one or more analyte sensors positioned one compartment from the convective transport forces of a fluid flow within the other compartment to allow reliable and accurate detection of cell-produced analytes and determination of the concentration of cell-produced analytes.

STATEMENT OF GOVERNMENT INTEREST

This invention was reduced to practice with Government support underGrant No. NIH/HL068164 awarded by the National Institutes of Health andGrant No. NSF/BES0301446 awarded by the National Science Foundation; theGovernment is therefore entitled to certain rights to this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a flow chamber and a method for detecting lowconcentrations of molecules produced by cells.

2. Description of the Related Technology

The flow conditions in conventional single compartment flow chamberspresent difficulties in detecting and measuring cell-produced diffusiblemolecules, which are typically produced in low concentrations.Diffusible molecules are rapidly carried from the cell surface into theflow current due to convective transport, forming a steep concentrationgradient that inhibits the accurate measurement of the diffused moleculeconcentration. The sensors utilized in conventional flow chambers arealso typically unable to make accurate measurements under flowconditions due to the sensor placement and poor flow signal sensitivity.Consequently, single compartment flow chambers are unable to accuratelyor reliably detect or measure the amount of diffusible moleculesproduced by cells contained within the single compartment flow chamber.

Specifically, conventional flow chamber systems are incapable ofaccurately detecting and measuring cell nitric oxide (NO) generated bycells placed under shear stress by contact with a flowing fluid. NO isknown to rapidly diffuse and has a short half-life of about 2-30seconds. Consequently, NO produces sharp gradients in concentration nearthe source of production due to convective transport which rapidlyremoves the NO that diffuses into the fluid from the cell surface. Thesteep concentration gradient and low concentration levels of NO producedby cells make accurate and reliable NO measurements under controlled invitro conditions virtually impossible. Although, placement of the NOsensing electrodes close to the exposed cell surface can reduce thepotential effects discussed above, such an electrode placement willcause disturbances in the flow profile in the vicinity of the cellsbeing monitored thereby altering the results by changing the effectiveshear stress due to the flow on some or all of the cells. Furthermore,the electrodes used for NO measurement, can also be sensitive to flow,thereby further distorting or masking the NO signal.

Research on shear stress-induced NO production has been severely limitedbecause of the foregoing experimental difficulties and detectionlimitations on concentration and accuracy. NO detection measured in asystem which applies changes in shear stress by changing the fluid flowis particularly challenging because of the low NO concentrations and themany competing phenomena including convection, diffusion, and chemicaldegradation. Similar problems exist in relation to measurement of othercell-generated species as well.

Consequently, there is a need to develop an improved flow chamber thataddresses the deficiencies of the prior art and enables detection andmeasurement of low concentration cell produced diffusible molecules,such as NO.

SUMMARY OF THE INVENTION

In a first aspect, the invention is directed to a flow chamber fordetecting an analyte. The flow chamber includes a first compartmenthaving a fluid inlet and fluid outlet for allowing a fluid to flowthrough the first compartment. The flow chamber further includes asecond compartment and an analyte sensor positioned within the secondcompartment. A permeable membrane, having a first surface that isexposed to fluid flow in the first compartment a second surfacepositioned within the second compartment separates the first and secondcompartments.

In a second aspect, the invention is directed to a flow chamber fordetecting an analyte. The flow chamber includes a first compartmenthaving a fluid inlet and fluid outlet for allowing a fluid to flowthrough the first compartment. The flow chamber further includes asecond compartment and an analyte sensor positioned within the secondcompartment. The flow chamber also includes a structure for allowingpassage of an analyte from the first compartment to the secondcompartment, wherein the analyte sensor is positioned at a knowndistance from a surface of said structure.

In a third aspect, the invention is directed to a method for detectingan analyte using a flow chamber described in the first aspect of theinvention. The method involves positioning a plurality of cells withinthe flow chamber on the first surface of the permeable membrane. Theanalyte sensor may be positioned a known distance from said plurality ofcells. Fluid is flowed through the first compartment, and the analyteproduced by said plurality of cells is detected.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross-sectional view of an exemplary flow chamber inaccordance with the present invention with the sensors located in thechamber.

FIG. 2 is a bottom view of a sensor holder for retaining an analytesensor in a sensing position without the sensors present.

FIG. 3 is a side view of the sensor holder of FIG. 2.

FIG. 4 is an exploded view of a parallel plate flow chamber including asensor holder without the sensors present.

FIG. 5 is a cross-sectional view the flow chamber of FIG. 4 with thesensors present.

FIG. 6 is a close-up cross-sectional view of a portion of FIG. 5 showingthe permeable membrane and the tips of the sensors.

FIG. 7 is a graph of the change in NO concentration as a function oftime showing shear stress-induced NO response corresponding to a changein shear stress from 0.1 dyn/cm² to 1, 6, 10 and 20 dyn/cm².

FIG. 8( a) is a graph of the change in NO concentration as a function ofthe shear stress change for experiments involving a change in shearstress from 0.1 dyn/cm² to 1, 6, and 20 dyn/cm².

FIG. 8( b) is a graph the time constant as a function of shear stresschange for experiments involving a change in shear stress from 0.1dyn/cm² to 1, 6, 10 and 20 dyn/cm²a.

FIG. 9( a) is a graph comparing the change in NO concentration as afunction of time showing shear stress-induced NO response to a stepchange from 0.1 to 1 dyn/cm² before and after treatment with 1 mML-NAME.

FIG. 9( b) is a graph comparing the change in NO concentration as afunction of time showing shear stress-induced NO response to a stepchange from 0.1 to 6 dyn/cm² before and after treatment with 1 mML-NAME.

FIG. 9( c) is a graph comparing the change in NO concentration as afunction of time showing shear stress-induced NO response to a stepchange from 0.1 to 10 dyn/cm² before and after treatment with 1 mML-NAME.

FIG. 9( d) is a graph comparing the change in NO concentrations asfunction of time showing shear stress-induced NO response to a stepchange from 0.1 to 20 dyn/cm² before and after treatment with 1 mML-NAME.

FIG. 10 is a graph of the change in NO concentration in response toshear stress step changes before and after treatment with L-NAME.

FIG. 11 is a graph of steady-state NO concentration as a function of therate of production of NO.

FIG. 12( a) is a graph comparing the experimental results for changes inshear stress (open bars, mean±SE) with steady-state change in NOconcentration predicted from 3 different models for shearstress-dependent rate of NO production (R_(NO)(τ): linear=black bars,hyperbolic=bars with horizontal hatching, sigmoidal=bars with diagonalhatching).

FIG. 12( b) is a graph showing NO production rates and corresponding NOrelease rates as a function of shear stress (r) with best-fit parametersfor each model.

FIG. 13 is a graph showing a comparison of the experimental results andmathematical simulations.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

For illustrative purposes, the principles of the present invention aredescribed by referencing various exemplary embodiments thereof. Althoughcertain embodiments of the invention are specifically described herein,one of ordinary skill in the art will readily recognize that the sameprinciples are equally applicable to, and can be employed in otherapparatuses and methods. Before explaining the disclosed embodiments ofthe present invention in detail, it is to be understood that theinvention is not limited in its application to the details of anyparticular embodiment shown. The terminology used herein is for thepurpose of description and not of limitation. Further, although certainmethods are described with reference to certain steps that are presentedherein in certain order, in many instances, these steps may be performedin any order as may be appreciated by one skilled in the art, and themethods are not limited to the particular arrangement of steps disclosedherein.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural references unless thecontext clearly dictates otherwise. Additionally, the terms “a” (or“an”), “one or more” and “at least one” can be used interchangeablyherein. It is also to be noted that the terms “comprising”, “including”,and “having” can be used interchangeably.

The present invention is directed to a flow chamber 100 and method fordetecting the presence of one more cell-produced analytes under flowconditions. Flow chamber 100 includes a first compartment 10 and asecond compartment 30 separated by a permeable membrane 50 on which aplurality of cells may be positioned. Permeable membrane 50 serves toinsulate one or more analyte sensors 70 positioned within secondcompartment 30 from the convective transport forces of fluid flow withinsecond compartment 30. Analyte sensor 70, located proximate to permeablemembrane 50, enables the detection of one or more cell-produced analytesthat diffuse through permeable membrane 50. Cell-produced analytes aregenerated in response to an applied stimulus and diffuse throughpermeable membrane 50 into second compartment 30 where the analytes aredetected by analyte sensor 70. In an exemplary embodiment, the inventionenables the determination of analyte concentration or a change inanalyte concentration over time.

As shown in FIG. 1, flow chamber 100 includes two compartments 10, 30and is arranged to detect the presence of one or more cell-producedanalytes generated in response to an applied stimulus. A firstcompartment 10 is configured as an enclosed structure defined by aceiling 12, floor 14 and plurality of side walls 16. First compartment10 further includes a fluid inlet port 18 and fluid outlet port 20through which a fluid may be flowed, for example, in order to applyshear stress to cells positioned therein. In one embodiment, a fluidsource is connected to fluid inlet 18 via a conduit and supplies a fluidto first compartment. The fluid is subsequently expelled via fluidoutlet port 20 and drained from the substantially closed system.Alternatively, fluid inlet port 18 and fluid outlet port 20 may beconnected via a conduit, forming a substantially closed system wherein afluid may be continuously circulated. In an exemplary embodiment, firstcompartment 10 forms a substantially controlled and closed system,wherein a pump is attached to and/or valves are positioned within fluidinlet port 18, fluid out let port 20, one or more conduits connectedthereto or combinations thereof in order to set and regulate thepressure within and prevent leakage of fluid from first compartment 10.

A second compartment 30 formed as a substantially enclosed or enclosedinterior space defined by a ceiling 32, floor 34 and plurality of sidewalls 36, may be configured as a closed system. In an exemplaryembodiment, second compartment 30 forms a substantially small, enclosedspace in order to ensure that the contents of second compartment 30 andthe diffused analyte quickly achieve equilibrium. In an exemplaryembodiment, second compartment 30 has a volume of about 1000 ml or less,preferably, about 500 μL to about 800 μL. Second compartment 30 islocated proximate to a permeable membrane 50 separating first and secondcompartments 10, 30. Due in part to the small volume and zero fluxcondition at the other boundaries of second compartment 30 and the shortdiffusion distance from permeable membrane 50, the concentration of ananalyte in second compartment 30 is substantially uniform and rapidlyequilibrates with the analyte concentration in the cell layer.Optionally, second compartment 30 may include a fluid inlet port 38 andfluid outlet port 40 that allows for a fluid to be introduced, containedand/or flowed through second compartment 30 for the purpose of rinsingsecond compartment 30 and its contents. In an exemplary embodiment,second compartment 30 may have the same flow system components andsystem configuration as that of first compartment 10.

Separating two or more compartments of flow chamber 100 is a permeablemembrane 50, which may be configured as any porous substrate thatprovides a surface on which a plurality of cells may be positioned. Inthe embodiment of FIG. 1, a plurality of cells are immobilized and/orcultured on a first surface 52 of permeable membrane 50 that is exposedto fluid flow within first compartment 10. For purposes of the presentinvention, any group of cells may be positioned on first surface 52 ofpermeable membrane 50, forming one or more cellular layers or a tissuematrix. Exemplary cells include cells that can adhere to or be culturedon a two dimensional substrate, such as epithelial cells, endothelialcells, vascular smooth muscle, fibroblasts, osteoblasts, chondrocytes,endothelial cells, including lymphatic endothelial cells, and stem cellsat various stages of differentiation.

Permeable membrane 50 allows for the passage of at least one analyte ofinterest from first compartment 10 to second compartment 30,particularly analytes produced by the cells positioned on permeablemembrane 50. Exemplary analytes include small molecules, preferablydissolved gases, such as nitric oxide (NO) and oxygen; ions such ascalcium, potassium, magnesium, hydrogen (pH); sugars, such as glucose,nucleotides such as adenosine triphosphate (ATP), adenosine diphosphate(ADP), and adenosine monophosphate (AMP); small proteins, such asendothelin; and lipids such as prostacyclin. Large molecule analytes,however, may also be investigated using flow chamber 100. In anexemplary embodiment, permeable membrane 50 has a plurality of aperturesor pores that allow for the diffusion of small analytes throughpermeable membrane 50. In an exemplary embodiment, the size of eachaperture or pore is from about 0.1 μm to about 12 μm in diameter,preferably, from about 0.1 μm to about 8 μm in diameter, morepreferably, from about 0.4 μm to about 5 μm in diameter and mostpreferably, from about 0.4 μm to about 3 μm in diameter. Additionally,the porosity, i.e. area fraction of the pores, of permeable membrane 50may be up to about 0.14, preferably, up to about 0.005 and morepreferably, from about 0.005 to about 0.5. The selected pore size andporosity of permeable membrane 50 is dependent upon the diffusibility ofthe analyte being investigated.

Positioned between first and second compartments 10, 30, permeablemembrane 50 is designed to shield the contents and environment of secondcompartment 30 from the convective forces generated by fluid flow infirst compartment 10. Specifically, the structure of permeable membrane50 substantially shields the cell-produced analytes that have diffusedacross permeable membrane 50, analyte sensor 70 and any fluid containedwithin first compartment 10 from the convective fluid flow within firstcompartment 10. This shielding effect is achieved by virtue of the factthat permeable membrane 50 forms a structural barrier that substantiallyinhibits the fluid flow, and consequently substantially inhibits orprevents the kinetic transfer of flow force, between the first andsecond compartments 10, 30.

As shown in the embodiment of FIG. 1, permeable membrane 50 separatesand forms a structural barrier between the first and second compartments10, 30. First surface 52 of permeable membrane 50 forms firstcompartment ceiling 12 and second surface 54 of permeable membrane 50forms second compartment floor 34. Permeable membrane 50 is preferablypositioned flush with respect to a wall of first compartment 10,allowing for a laminar and uniform fluid flow in first compartment 10.Alternatively, permeable membrane 50 may be positioned so as to protrudeor be recessed within first compartment 10, thereby causing a morecomplex and possibly turbulent fluid flow pattern in first compartment10 due to interference of the permeable membrane 50 with the fluid flow.As shown in FIG. 1, permeable membrane 50 is arranged so as to separateflow chamber 100 into a first compartment 10 and a second compartment30. One of ordinary skill in the art, however, would appreciate that solong as permeable membrane 50 creates a structural barrier between twoor more compartments of flow chamber 100, alternative flow chamberconfigurations may be utilized. For example, first and secondcompartments 10, 30 may be arranged in a side-by-side orientation,wherein permeable membrane 50 forms side walls 16, 36 of first andsecond compartments 10, 30.

One or more analyte sensors 70, positioned within second compartment 30,can be used to detect the presence of an analyte, determine analyteconcentration, determine a change in analyte concentration, determineanalyte production rate or combinations thereof. In one embodiment, twoor more analyte sensors 70 may be positioned within second compartment30, wherein each analyte sensor 70 is customized to detect a differentanalyte. Alternatively, a plurality of the same analyte sensors 70 canbe positioned at different locations within second compartment 30. Thisembodiment may be useful, for example, for studying the effects ofcomplex flow patterns on the generation of analytes by cells since itcould give information about analyte generation at different locationsalong the immobilized cells exposed to a stimulus, such as a fluid flowin first compartment 10. Analyte sensor 70 may be any sensor suitablefor detecting an analyte, such as an electrode or piezoelectriccantilever sensor, and may be capable of detecting an analyte under dryor wet environmental medium. Exemplary analyte sensors 70 may includehigh sensitivity sensors capable of detecting small molecules,preferably dissolved gases, such as nitric oxide (NO) and oxygen; ionssuch as calcium, potassium, magnesium, hydrogen (pH); sugars, such asglucose, nucleotides such as ATP, ADP, and AMP; small proteins, such asendothelin; and lipids such as prostacyclin.

In order to quantify the concentration or change in concentration of oneor more analytes produced by the cells positioned on permeable membrane50, analyte sensor 70 may be placed within second compartment 30 at aknown, fixed distance from the cells, which can be approximated by thedistance to first surface 52 of permeable membrane 50. Alternatively,second compartment 30 can be fabricated to be sufficiently small thatfluid contained in second compartment 30 will quickly equilibrate withcell-generated analyte that diffuses through permeable membrane 50 toprovide quantitative information about analyte concentration. Analytesensor 70 may be fixed to, or located proximate to, any surface ofsecond compartment 30, including ceiling 12, floor 14, side walls 16, orcombinations thereof. Alternatively, analyte sensor 70 may be removablyinserted into second compartment 30 via an analyte sensor port 72. Thedistance between analyte sensor 70 and the cell surface/first exposedsurface 52 maybe adjusted and fixed by virtue of fasteners, such asclamps and threaded fasteners. In an exemplary embodiment, secondcompartment 30 may be constructed from a substantially transparentmaterial having a plurality of graduated indicators on a surface thereofthat provides a guide and means for measuring the distance betweenanalyte sensor 70 and first surface 52 of membrane 50 on which the cellsare located.

In the embodiments shown in FIGS. 2-4, flow chamber 100 further includesa sensor holder 80 that can be associated with second compartment 30 andanalyte sensors 70 to facilitate positioning analyte sensors 70 in afixed, predetermined distance relative to the first surface 52 ofpermeable membrane 50. Sensor holder 80, having the interior spacedefining and second compartment 30, is inserted into a sleeve 82configured as a container with a shape, size and dimensions thatcorresponds to and is adapted to securely receive a portion of sensorholder 80, preferably the interior space defining second compartment 30and second compartment 30. In this embodiment, permeable membrane 50forms a lower surface 84 of sleeve 82. Preferably, the distance betweena lower surface of sensor holder 80 and permeable membrane 50 is about300 μm. Sleeve 82 is adapted to contain one or more liquids, providing asensing medium in which analyte sensor 70 is bathed upon insertion intosleeve 82. Prior to insertion into sleeve 82, floor 34 is absent fromsecond compartment 30, creating an open system wherein a distal end ofsensor 70 is unconfined. Optionally, one or more micromanipulators 86may be attached to sensor holder 80 in order to allow a user to interactwith sensor holder 80, second compartment 30, analyte sensor 70 or acombination thereof when flow chamber 100 is placed under a microscope.

To facilitate the detection of analytes present in low concentration,analyte sensors 70 can be positioned proximate to second surface 52 ofpermeable membrane 50. Preferably, analyte sensor 70 may directlycontact second surface 52 of permeable membrane. In an exemplaryembodiment, analyte sensor 70 is positioned about 5 μm to about 50 μm,preferably, about 5 μm to about 20 μm, from a second surface 52 ofmembrane 50. The selected distance of analyte sensor 70 relative toexposed surface 52 may be dependent upon the size and concentration ofthe analyte being investigated. The purpose of accurately positioningthe analyte sensor 70 is that it allows calculation of analyteconcentrations from known concentration gradients in case it isdesirable to determine analyte concentrations in this manner.

In addition to analyte sensors 70, flow chamber 100 may include one ormore environmental sensors 74 for monitoring one or more environmentalconditions of one or more compartments of flow chamber 100. Exemplaryenvironmental sensors 74 that may be positioned within first or secondcompartment 10, 30 may include temperature sensors, pressure sensors,flow rate sensors or combinations thereof. Environmental sensors 74 maybe fixed to a surface of, or removably inserted via an environmentalsensor port 76 into, a flow chamber compartment 10, 30. In an exemplaryembodiment, environmental sensors 74 may be connected to a systemcapable of adjusting an environmental condition of a flow chambercompartment in response to measurements obtained from one or moreenvironmental sensors 74.

During operation, a plurality of cells may be immobilized and/orcultured on first surface 52 of permeable membrane 50. While a pluralityof cells may be positioned on first surface 52 forming multiple celllayers that extend into the interior of first compartment 10, the cellsadjacent to the first surface 52 of membrane 50 are located at a knowndistance relative to analyte sensor 70 since this distance can bedetermined by summing the thickness of membrane 50 with the distancefrom analyte sensor 50 to second surface 54 of membrane 50.Specifically, as shown in FIG. 1, the cells directly contact and arealigned along first surface 52. Consequently, the distance betweensensor 70 and the plurality of cells may be approximated by the distancebetween sensor 70 and first surface 52.

A stimulus is then applied to the cells, inducing the cells to generateor change the production of one or more analytes. In an exemplaryembodiment, the applied stimulus may be shear stress exerted on thecells by a fluid flow within first compartment 10; the addition orwithdrawal of stimulating or inhibiting molecules under steady fluidflow conditions in first compartment 10; environmental changes, such aschanges in temperature, pressure or flow rate, in first compartment 10;or combinations thereof.

In one embodiment, a fluid is flowed through first compartment 10 in adirection substantially parallel to ceiling 12, forming a laminar flowthat applies a substantially uniform shear stress to the cells. Theapplied shear stress induces the cells to produce or modify theproduction of one or more analytes. In an exemplary embodiment, thephysiological shear stress applied to the cells is about 0 to about 200dyn/cm². It is desirable to achieve the shear stress with the lowestflow rates possible in order to limit the convective transport of theanalyte. This may be accomplished by minimizing the cross-sectionaldimensions of the flow chamber subject to practical limitations anddesired uniformity of the flow field.

Alternatively, the fluid flow may have a more complex and/or turbulentflow pattern that may be achieved using a variety of different methods.In one embodiment, a complex flow pattern may be created by virtue ofthe placement of permeable membrane 50 relative to the fluid flow. Inanother embodiment, the geometry of flow chamber 100 or one or morecomponents thereof can be configured or modified so as to produce anynumber of flow patterns of interest. For example, a sudden expansion infirst compartment 10 would produce flow separation creating arecirculation zone that mimics flow at branch points in blood vessels.Also, the flow pattern may be modified by fluctuation of the fluid flowrate. In addition to incremental step changes in flow rate, any numberof time-dependent flow patterns including periodic waveforms could beused.

The cell-produced analytes that are generated in response to thestimulated cells diffuse through permeable membrane 50 into secondcompartment 30. By virtue of the small, substantially closed andstagnant fluid environment, of second compartment 30, the fluid andanalyte within second compartment 30 can be induced to quickly reachequilibrium. Additionally, permeable membrane 50 provides a structuralbarrier that insulates the diffused analytes from the effect ofconvective transport due to fluid flow in first compartment 10, ensuringthat the measurements made by one or more analyte sensors 70 positionedwithin second compartment 30 accurately reflect the analyteconcentration produced by the cells. These features enable flow chamber100 to accurately and reliably detect and/or measure the concentrationof analytes present in low concentrations.

In order to quantify analyte concentration without having to allow thecontents of second compartment 30 to equilibrate and/or detect a changein analyte concentration over time within second compartment 30, analytesensor 70 may be fixed or removably positioned within second compartment30 at a known distance relative to the first surface 52 of membrane 50on which the cells are located. Preferably, a distal end of analytesensor 70 contacts second surface 54 of membrane 50 to establish adistance between distal end of analyte sensor 70 and first surface 52 ofmembrane 50 equal to the membrane thickness.

In the embodiment shown in FIGS. 2-4, second compartment 30 and analytesensor 70 may be attached to a sensor holder 80 and bathed within aliquid bath contained within sleeve 82 to enhance detection sensitivity.Sensor holder 80, which has an interior space defining secondcompartment 30, is subsequently inserted into sleeve 82, containing afluid bath in which analyte sensor 10 is bathed. Flow cell 100 may beplaced on the platform of a microscope and sensor holder 80, secondcompartment 30, analyte sensor 70, or combinations thereof may bemanipulated using micromanipulators 86.

Upon fixing the distance between sensor 70 and first surface 52 ofmembrane 50, it may be possible to determine the change in analyteconcentration over a predetermined interval of time by comparingmeasurements taken by analyte sensor 70 at two different predeterminedflow rates or by employing more complicated flow patterns which, forexample, vary over time. Analyte concentration within second compartment30 may also be determined at a predetermined point by calibratinganalyte sensor 70, for example, by flushing second compartment 30 withan experimental fluid to obtain a baseline value for a particularanalyte.

To calibrate sensor 70 and enhance the accuracy of the measurementstaken by sensor 70, second compartment 30 and analyte sensor 70positioned therein may be periodically rinsed by flushing a liquid, suchas distilled water or cleaning solution, through second compartment 30,said fluid entering via inlet port 38 and exiting at outlet port 40.Additionally, analyte sensor 70 may be periodically removed from flowchamber 100 for cleaning between measurements.

To further ensure optimal sensing conditions, environmental sensors 74may be positioned within first and/or second compartment 10, 30 tomonitor an environmental condition of the compartment, such as pressure,temperature and fluid flow rate. A system, operatively associated withenvironmental sensors 74, may automatically adjust and regulate one ormore environmental conditions of the flow chamber 100 in response to ameasurement obtained from one or more environmental sensors 74. Forexample, the temperature within second compartment 30 may be adjusted tooptimize detection. Additionally, the pressure in first and secondcompartments 10, 30 may be regulated to provide a substantially neutralpressure flow chamber 100 system.

The measured analyte concentration is dependent on the analyteproduction rate as well as the convective and diffusive mass transportprocesses occurring in the flow chamber. The flow chamber may becalibrated to account for the mass transport effects. Alternatively, itmay be possible to determine the dynamic change in NO production byusing mathematical modeling, such as the model described in A. A. Fadel,K. A. Barbee, D. Jaron, “A computational model of nitric oxideproduction and transport in a parallel plate flow chamber”, Ann. BiomedEng. 37 (2009) 943-954, which is incorporated by reference herein in itsentirety. The mathematical model may be used to relate the steady-stateNO concentration at the position of the electrode to the analyteproduction rate.

Additionally, the mathematical model may be used to determine theinitial analyte production rate, which may be estimated by fitting atheoretical mathematical function to the relationship betweensteady-state analyte concentration changes for a range of shear stressstep changes. In an exemplary embodiment, this relationship may bemathematically modeled using a simple linear model or a nonlinearrelationship model, such as a hyperbolic model or sigmoidal model. Themathematical model enables the determination of steady-state analytevalues at various applied shear stresses, allowing the steady statedifference in the change in NO concentration between any two shearstress levels to be determined. This procedure is described in A.Andrews, D. Jaron, D. Buerk, P. Kirby and K. Barbee, “Direct, real-timemeasurement of shear stress-induced nitric oxide produced fromendothelial cells in vitro” Nitric Oxide, 23 (2010) 335-342,incorporated by reference herein in its entirety.

The innovative flow chamber 100 offers a unique advantage by providingreal-time, direct, spatial and temporal in vitro detection of analytespresent in low concentrations. By insulating the analyte in a stagnantsecond compartment 30 separated by permeable membrane 50 from convectivetransport due to fluid flow in first compartment 10, fluid system 100prevents disturbances in the fluid flow profile, prevents possibleeffects of the flow on analyte sensor 70, and avoids problems associatedwith chamber leaking at the sensor insertion site. Additionally bycontrolling the distance of analyte sensor 70 from the cell surface andor configuring second compartment 30 as a substantially small andenclosed space to ensure rapid equilibration and analyte and fluid insecond compartment 30, accurate, and reliable quantitative concentrationmeasurements can be obtained. Additionally, the use of the flow chamberto conduct in vitro experiments offers a significant advantage byproviding the ability to control shear stress and determine analyteconcentration changes in real time. This is due, in part, to the factthat the measured analyte concentrations reflect a combination ofanalyte production by the cells and convective and diffusive masstransport effects of the system. Experimental data may be coupled withmathematical modeling to interpret the results and to relate analyteproduction to shear stress.

Flow chamber 100 of the present invention may be used for variety ofapplications and may be particularly well suited for use as a researchtool. In an exemplary embodiment, flow chamber 100 may be used tomeasure the amount of or changes in small cell-produced molecules, suchas NO, generated in response to an applied fluid flow shear stress. Inone embodiment, the flow chamber can be used to conduct and evaluatesimulations in which specific signaling events are explicitly modeled.The signaling pathways can be studied in detail by simulating theeffects of inhibitors or other interventions and comparing the effectson the dynamics of the NO response. Therefore the flow chamber may beused to investigate the mechanisms that determine NO production. Theinvention may further provide a method for studying the kinetics of thesignaling mechanisms linking NO production with shear stress as well aspathological conditions involving changes in NO production oravailability. Additionally, the flow chamber may be used to evaluatedifferent theoretical models which have previously been limited by thepaucity of quantitative data regarding production and transport of NO.

The device can also be used to detect and study other cell-produceddiffusible molecules present in low concentrations. Alternatively, flowsystem 100 may be used for chemical analyte analysis that and need notinvolve immobilizing cells on permeable substrate 50 or generatingcell-produced analytes.

EXAMPLES Example 1

In an experimental study, direct, real-time measurement of NOconcentration changes due to flow-induced shear stress stimulation ofendothelial cells in vitro using the flow chamber of the presentinvention was investigated. The measured NO concentrations reflect acombination of NO production by the cells and convective and diffusivemass transport effects of the system. The experimental protocol setforth in the study provides a method for studying the mechanisms linkingNO production with shear stress as well as pathological conditionsinvolving changes in NO production or availability.

Bovine aortic endothelial cells (BAECs) were cultured in Dulbecco'smodified Eagle's medium (Mediatech Cellgro), supplemented with 10% fetalbovine serum (Sigma), 2 mmol/l L-glutamine (Mediatech Cellgro), andpenicillin-streptomycin (Mediatech Cellgro). The cells were grown toconfluency and subsequently plated onto the lower surface of individualpolyester Transwell® membranes (Corning Transwell Permeable Supportshaving a 24 mm diameter culture area with 3 μm pores). The Transwell®membranes were then placed in an incubator overnight, inverted andcultured for 1 day before conducting the experimental study. TheTranswell® membranes were subsequently washed 3 three times with asolution of Dulbecco's Phosphate Buffered Saline (PBS) withcalcium/magnesium (Sigma) supplemented with 70 μM L-arginine (L-arg)(Sigma) and inserted into the flow chamber.

The flow chamber included an electrode and equipment for measuring NO(TBR4100 4-channel Free Radical Analyzer and 200 μm diameter minisensors for NO measurements ISO-NOPF). The electrodes were frequentlyrecoated with nafion (Sigma) and re-calibrated during the experimentalstudy to improve selectivity. The recoating process involved at leasttwo dip/dry sessions. The electrodes were calibrated by thedecomposition of a NO donor S-nitroso-N-acetyl-penicillamine (SNAP)using Cu(II). The SNAP was prepared by dissolving 5 mg EDTA and 5.0mg+/−2.0 mg of SNAP in 250 mL HPLC grade water. The electrode wasimmersed in 12 mL of 0.1 M copper(II) sulfate in distilled water forabout 1 to about 2 hours until the electrode stabilized. Aliquots ofSNAP were sequentially added in an amount of about 10 μL, 20 μL, 40 μL,80 μL after each signal reached a plateau. A multipoint calibration plotwas created using Data-Trax software (produced by WPI). The samplingrate was 10 samples/s. The change in recorded potential was converted tocorresponding molarities of NO produced by SNAP addition. The efficiencyof the conversion of SNAP to NO was 0.6. Electrode sensitivity was atleast about 10 pA/nM. The electrodes were tested for sensitivity tonitrite (NO₂ ⁻) after the calibration procedure and sensitivity wasfound to be about 1.5% to about 3% per 100 mM. Sensitivity increasedwhen the electrode sensitivity to NO had decreased significantly below 5pA/nM. The temperature probe supplied by WPI was pre-scaled using atwo-point entry of known temperatures (0.03125V=1° C., 0.625V=20° C.).

As shown in FIG. 4, the flow chamber was made from parallel plates ofpolycarbonate with a spacer that determined its height. The dimensions(in cm) of the flow channel were about: 4.57 W×12.19 L×0.025 H. The flowinlet and outlet had large reservoirs with sampling ports. A glasscoverslip covered an opening in the bottom plate, allowing visualizationof the cell layer and the electrode on an inverted microscope. As shownin FIGS. 5-6, endothelial cells were grown on the lower surface of aTranswell® porous membrane, which fit into the flow chamber flush withthe upper plate of the flow chamber. The flow chamber had an uppercompartment and lower compartment separated by the porous Transwell®membrane. Below the Transwell® membrane, fluid flowed through theparallel plate channel that defined the lower compartment, exposing theendothelial cells to uniform shear stress. Above the Transwell®membrane, a stagnant upper fluid compartment, having a small volumehoused the NO electrode and temperature sensor. This design placed theelectrode out of the fluid flow avoiding problems associated withpotential flow sensitivities, flow disturbances and chamber leaking atthe electrode insertion site. Furthermore, due to the small volume andzero flux condition at the other boundaries of the stagnant upper fluidcompartment and the short diffusion distance through the membrane, theconcentration of NO in the upper fluid compartment was nearly uniformand rapidly equilibrated with the concentration in the cell layer. Theelectrode was lowered into the upper compartment of the flow chamberuntil it rested on the surface of the Transwell® membrane at a fixeddistance from the ECs of about 10 μm equal to the thickness of themembrane. This configuration allowed the NO generated by the cells to bemeasured abluminally from the endothelial cell layer. Due to thetemperature sensitivity of the NO electrode, the flow chamber wasenclosed in a water bath at 37° C. in order to prevent heat loss andtemperature fluctuations. In addition, samples were taken duringexperiments from sampling ports and later analyzed for NO concentrationusing an NO Analyzer (Sievers NA0280i).

The flow chamber was sterilized under ultraviolet light for 20 minutesbefore each use. The flow chamber was flushed with each of thefollowing: 100 mL of 70% ethanol and 100 mL of deionized water and thenprepped with 75 mL of the PBS with calcium and magnesium supplementedwith 70 μL-arg solution. The PBS with calcium and magnesium supplementedwith 70 μM L-arg solution was pumped into the lower compartment of theflow chamber using a Reglo-Z Digital pump (Ismatec). Flow rates werecalculated based on desired shear stresses using Equation 1.

Q=τwh ²/6μ  Equation 1

where w is the chamber width of the lower flow chamber, h is the chamberheight of the lower flow chamber, μ is the viscosity, τ is the shearstress (dyn/cm²) and Q is the flow rate. The pump was controlled using aLabView program, which was adapted from the manufacture's online LabViewdriver. Using an inverted light microscope (Nikon TE300 Eclipse) under10× objective, the electrode was lowered using a micrometer until theelectrode gently rested on the surface of the Transwell® membrane. Thechamber was then placed in an enclosed heated water bath of about 37° C.for an hour without flow until the electrode and temperature in the flowchamber stabilized.

120 mL of the PBS with calcium and magnesium supplemented with 70 μML-arg solution was cycled continuously through the lower compartment ofthe flow chamber during the experiment at a low flow rate of 0.25 mL/min(corresponding to a wall shear stress of 0.1 dyn/cm²) to prevent theaccumulation of NO due to basal (unstimulated) production. The cellsplated on the Transwell® membrane were exposed to multiple step changesin flow rate ranging from about 0.1 dyn/cm² to about 20 dyn/cm² with a3-minute interval between the step changes. Specifically, as shown inFIG. 7, a series of step changes in flow rate corresponding to shearstresses of about 1, 6, 10, and 20 dyn/cm² were applied, alwaysreturning to 0.1 dyn/cm² between stimuli.

The PBS with calcium and magnesium supplemented with 70 μM L-argsolution was then exchanged with PBS with calcium/magnesium with 1 mMNω-Nitro-L-arginine methyl ester (L-NAME, pH 7.2, Sigma). The L-NAMEsolution was flushed through the lower compartment of the flow chamber.Fluid flow was subsequently turned off for 1 hour prior to repeating thesame sequence of step changes that were performed prior to L-NAMEtreatment.

The step changes in flow elicited reproducible changes in NOconcentration, wherein cells could be repeatedly stimulated withoutdiminution of the response. The magnitudes of the responses wereconsistent within an experiment but varied among cultures. The chamberwas continuously perfused at a low flow rate of 0.25 mL/min(corresponding to a wall shear stress of 0.1 dyn/cm²) to prevent theaccumulation of NO due to the basal (unstimulated) NO production. Aseries of step changes in flow rate corresponding to shear stresses of1, 6, 10 or 20 dyn/cm² were applied, always returning to 0.1 dyn/cm²between stimuli. The steady-state NO concentration at 0.1 dyn/cm² wasoffset to zero in order to show the change in NO concentration inresponse to each step change in shear stress, shown to occur at 50second intervals. The cellular response to the applied shear stressinvolved a sharp, transient decrease in NO concentration upon stepchange initiation followed by an increase in NO concentration to a new,higher steady-state.

The change in steady-state was calculated as the difference between thebaseline prior to the step change and the steady-state value followingthe step change, as shown in FIG. 8( a). The differences were calculatedusing the average concentration over a 13 s interval prior to the stepchange and the average concentration at the new steady-state over a 9 sinterval after the step change. The steady-state change averaged about−6 nM for a step change to 1 dyn/cm², about 25 nm for a step change to 6or 10 dyn/cm² and about 45 nM for a step change to 20 dyn/cm². Inaddition, the magnitude of the initial decrease was found to beshear-stress dependent. For the steady-state concentration to increasewith increased shear stress, the NO production rate must exceed the rateof removal by the increased convective transport effects.

The steady-state change at 20 dyn/cm² was statistically different fromthe change observed in response to 6 or 10 dyn/cm² but was notstatistically significant between 6 and 10 dyn/cm². The steady-statechange from 0.1 to 1 dyn/cm² was statistically different from stepchanges to all the other shear stresses. Concentration changes rangedfrom about −21 nM to about 9 nM, from about 19 nM to about 53 nM, fromabout 20 nM to about 47 nM and from about 24 nM to about 62 nM for astep change to 1, 6, 10 and 20 dyn/cm². However, because convectivetransport is higher at the higher flow rate, the NO production rate musthave been higher at 10 dyn/cm² than at 6 dyn/cm². Additionally, themagnitude of the initial decrease in NO concentration was found to beshear-stress dependent, suggesting that the transient decrease wasrelated to the convective effect of the step change in flow.

As shown in FIG. 8( b), the time course of the NO concentration profilesfollowing a step change were analyzed using an exponential fit tocalculate the time constant (t_(c)). Average t_(c) values were 64 s, 41s, and 21 s for 6, 10 and 20 dyn/cm², respectively. Exponential curvesdid not accurately reflect the time course for a step change from 0.1 to1 dyn/cm². Mean and SE were plotted and statistics were calculated usingthe paired two-tail t-test n=8 for 1, 6 and 10 dyn/cm² and n=6 for 20dyn/cm², p<0.05. For time constants, one value each for 6 dyn/cm² andfor 10 dyn/cm² were significant outliers and were excluded using Grubb'stest α=0.05. The ability to measure changes in NO concentration inreal-time allowed for analysis of the kinetics of the responses ofendothelial cells to changes in shear stress. The time constantscharacterizing the rate at which the concentration approached thesteady-state decreased significantly as the size of the step change inshear stress increased. This suggests that in addition to thesteady-state production rate being dependent on shear stress, the rateat which the signaling processes leading to increased production areactivated is also dependent on the size of the shear stress stimulus.The simulation of the time course of NO concentration changes due to aninstantaneous increase in production in response to a step in shearstress indicates that there is negligible transport lag in themeasurement system. Therefore, the time dependence of the concentrationchanges reflects the dynamics of the cellular response.

Shear stress-induced NO responses were compared before and after L-NAMEtreatment. Following stimulation at multiple shear stresses, theexperimental fluid was exchanged with 1 mM L-NAME having a pH of 7.2.Measurements with L-NAME were made under the same protocols as were usedprior to treatment with L-NAME with the shear stress starting at 0.1dyn/cm² and then increasing in step changes to 1, 6, 10 or 20 dyn/cm².As shown in the sample traces of FIGS. 9( a)-9(d), step change occurs at50 second intervals. The steady-state concentration at 0.1 dyn/cm² wasoffset to zero in order to show the individual NO response due to thestep change. The solid lines in these figures represent the NO responseprior to L-NAME treatment and the dotted line represents the NO responseafter L-NAME treatment, with the exception of the range of 0.1 to 1dyn/cm².

FIG. 10 shows a comparison of the steady-state NO concentration changesin response to a step change before and after treatment with L-NAME.These changes were calculated between steady-state concentrations beforeand after application of a step change in shear stress. Comparison ofthe steady-state changes between untreated and L-NAME treated responseswere found to be statistically significant between step changes from 0.1to 6, 10 and 20 dyn/cm² but not for 0.1 to 1 dyn/cm² but were not for0.1 to 1 dyn/cm². FIG. 10 shows that steady-state changes aftertreatment with L-NAME were reduced by 40% and were statisticallysignificant for all changes in shear stress, with p values of p<0.05 andp<0.01 for a paired one-tailed t-test. The mean and SE were plotted,wherein n=8 for 6 and 10 dyn/cm², n=6 for 20 dyn/cm², p<0.05; p<0.01;for paired one-tailed t-test. Steady-state changes after treatment withL-NAME averaged 60% of the untreated values. One value for 6 dyn/cm² wasa significant outlier and was excluded using Grubb's test α=0.01. Theconcentration changes were attenuated by the endothelial nitric oxidesynthase (eNOS) inhibitor L-NAME, confirming the validity of the flowchamber technique for NO measurement. A decrease in the baselineconcentration following treatment with L-NAME was observed but was notreflected in the data because the measurements only examine relativechanges in NO concentration. Thus, the effects of L-NAME treatment onthe absolute concentrations of NO were more profound than suggested bythe 40% reduction of the shear stress response.

The measured NO concentration is dependent on the production rate of NOas well as the convective and diffusive mass transport processesoccurring in the flow chamber. To account for the transport effects inthe flow chamber and consequently, properly interpret the measuredconcentration changes, the transport process was mathematical modeled todetermine the dynamic changes in NO production. The experimental resultswere compared to a mathematical model of transient and steady-state NOtransport. A. A. Fadel, K. A. Barbee, D. Jaron, “A computational modelof nitric oxide production and transport in a parallel plate flowchamber”, Ann. Biomed Eng. 37 (2009) 943-954, describes the developmentof the mathematical model. Within the flow domain (described by planePoiseuille flow), the convection-diffusion equations for the transportof NO were solved using finite element analysis. Production of NOoccurred in the 5 um thick endothelial layer where mass transport is bydiffusion only. An auto-oxidation reaction was included in both domainswith the oxygen concentration taken to be a constant throughout thechamber. The dimensions and configurations of the chamber in themathematical computations accounted for a membrane of about 10 μm andthe stagnant compartment above the cell layer.

The model permitted relation of the steady-state NO concentration at theposition of the electrode to the production rate within the endotheliallayer. This relationship was approximately linear with the slopestrongly dependent of the shear rate, as shown in FIG. 11. The solidlines of FIG. 11 represent the relationship of steady-state NOconcentration as a function of R_(NO) for each of the shear stressvalues used in the study. These relationships demonstrated that the sizeof a stimulated change in production rate, represented by the measuredchange in NO concentration depends on the baseline concentration.

Although the measurement technique used in this experiment onlydetermined the relative changes in NO concentration, using thecomputational model for the flow chamber, it was possible to estimatethe basal production rate of NO based on an analysis of the steady-stateconcentration changes for a range of shear stress step changes. If thebaseline concentration at 0.1 dyn/cm² is known or can be estimated, thenthe measured change in NO concentration can be related to the change inproduction rate. By fitting an expression for the shear-stress dependentproduction of NO to the measured changes in NO concentration in responseto a range of shear stress steps, the basal production rate wasestimated. Three relationships for shear-stress dependent R_(NO) wereinvestigated to determine the best fit for the steady-state experimentalresults for each relationship, as shown in FIGS. 12( a)-12(b).

The simplest model tested for NO production rate was linear with shearstress (R_(NO)=R_(basal)+Aτ; black bars), where R_(basal) is the basalproduction rate, R_(NO) is the rate of NO production, A is the slope andt is shear stress. Two nonlinear relationships for R_(NO) as a functionof shear stress were also investigated: a hyperbolic model(R_(NO)=R_(basal)+R_(sat)τ/(τ+A); represented in FIG. 12( a) by the barwith horizontal hatching, where R_(sat) is the maximum rate ofstimulated production (above R_(basal)), and a sigmoidal model(R_(NO)=R_(max)/(1+A exp^(−Bτ)); hatched bars), where R_(max) is themaximum rate of production. The basal rate for the sigmoidal model canbe calculated from R_(basal)=R_(max)/(1+A). Predicted steady-state NOvalues at each shear stress for each relationship were obtained from thecomputational model for the flow chamber, allowing the steady-statedifference in the change in NO concentration between any two shearstress levels to be determined.

Comparisons for the best fit of these models to the experimental data(mean±SE, open bars) are shown in FIG. 12( a). The linear model(represented by the black bar) provided the least suitable fit. The twononlinear models appear to provide excellent fits to most of theexperimental data, although both underestimate the change in NO for thechange in shear stress from 0.1 to 6 dyn/cm². The hyperbolic model(represented by the bar with horizontal hatching) provided the closestmatch to the data. Although the sigmoidal function could be made to fitthe discrete data points fairly well, the production rate was nearlyconstant within the plateau phase of the curve while shear stress (andthus convective transport) increased. This would lead to anon-physiological situation in which NO concentration decreases at shearstress values higher than a local maximum occurring between 10 and 20dyn/cm². Calculated values for the basal NO production rate (R_(basal))at zero shear stress and R_(NO) at different shear rates based onparameters that provided the best fit to the to the experimental datafor each relationship are summarized in Table 1 and shown graphically inFIG. 12( b). In FIG. 12( b), the calculated R_(NO) and release rates forexperimental shear stresses are shown for the hyperbolic model at τ=1dyn/cm².

TABLE 1 Analysis of experimental results from flow chamber usingdifferent models of shear-stress dependent NO production. τ (dyn/cm²) =0 0.1 1 6 10 20 Model R_(basal) R_(NO) (τ) (nM/s) R_(NO) = R_(basal) +Aτ 1.74 2.49 9.26 48.9 76.9 152 R_(NO) = R_(basal) + R_(max) τ/(τ + A)2.13 3.43 14.8 69.1 104 168 R_(NO) = R_(max)/(1 + A exp^(−Bτ)) 1.12 1.171.83 19.3 73.0 129 Model parameters. Linear, A = 7.52 (nM/s)/(dyn/cm²).Hyperbolic, R_(max) = 457.5 nM/s, A = 35 dyn/cm². Sigmoidal, R_(max) =129.5 nM/s, A = 115, B = 0.5 cm²/dyn.

The three models for shear stress-dependent R_(NO) were also fit tosteady-state NO concentration change data that was obtained from theL-NAME studies. The hyperbolic model provided the best fit, and thelinear model the least suitable fit. All the models underestimated theexperimental data for the 0.1 to 1 dyn/cm² change in shear stress. Theanalysis found that L-NAME partially inhibited R_(NO) under theexperimental condition of the study (L-arg also in perfusate). At thehighest shear stress change (0.1 to 20 dyn/cm²), R_(NO) was about 57.3%,about 57.6%, and about 58.3% of R_(NO) estimated from untreated ECsusing the linear, hyperbolic and sigmoidal models, respectively.

The computer models also permitted investigation of the role of masstransport in the dynamics of the transient response. As shown in FIG.13, simulations of NO concentrations were evaluated at the electrodelocation for a step change in shear stress from about 0.1 dyn/cm² toabout 20 dyn/cm². The initial decrease in NO concentration was followedby an increase to an elevated steady-state concentration. Twomathematical simulations are shown in FIG. 13 utilizing either atime-dependent (ramp) (dashed line) or time independent (instantaneousstep) relationship (dotted line) between NO production and shear stress.Simulations were performed for a shear stress change from 0.1 to 20dyn/cm², which occurred at 50 s. The time-independent model demonstratedthat following a step change a steady-state concentration was reachedalmost instantaneously. The time-dependent model utilized a linearlyincreasing production rate in response to the initiation of shearstress. This produced an initial decrease in NO concentration inresponse to the change followed by an increase until a new steady-stateconcentration was reached. The model describes NO production as a basalproduction rate plus a shear stress-dependent NO production component.In one simulation, the stimulated production rate of NO was dependent onshear stress alone, with no lag between change in shear stress and theincrease in production rate. In this case, the NO concentration reachedits new steady-state almost instantaneously. In contrast, when a gradualincrease in production rate linear with time was simulated, it waspossible to mimic the time-dependent changes in NO concentrationobserved experimentally, including the transient decrease and subsequentslower rise to the new steady-state. The nearly instantaneous responseto a step change in production indicates that the response time of theNO measurement is not limited by diffusion through the membrane. Thetime course of the NO concentration changes therefore reflects atime-dependent cellular response rather than a transport lag.

The experiment enabled direct, real-time measurement of NO concentrationchanges due to flow-induced shear stress stimulation of endothelialcells in vitro. The concentration changes were partially attenuated bythe endothelial nitric oxide synthase (eNOS) inhibitor L-NAME. Thefailure of L-NAME to completely abolish the NO response may in part bedue to the incomplete removal of L-arg from the upper compartment.However, the level of inhibition that was observed is consistent withprevious studies with L-NAME treatment of in vivo vessels.

The experimental results indicated that following a step change from alow shear stress of 0.1 dyn/cm² to higher shear stresses, the NOconcentration at the electrode first transiently decreased and thenincreased to a steady-state concentration that is higher than theinitial steady-state value except for a shear stress change to 1dyn/cm². As suggested by the experimental simulations, this initialdecrease is due to convective washout whose effect is immediate. Sincethe simulated production rate is actually the net release of NO from thecell (production minus any consumption by the cell), in this experiment,changes in NO due to NO production were indistinguishable from changesin NO due to reactions consuming NO within the cell. Thus, thepossibility that the lag in release was due in part to increasedproduction accompanied by a simultaneous increase in NO consumptionthrough rapid reactions with reactive species such as superoxide orlipid peroxyl radicals could not be ruled out. When an instantaneousincrease in production rate was simulated, it was predicted that a veryrapid increase in NO concentration with no transient decrease would beobserved. In addition, the magnitude of the initial decrease in theexperimental results was found to be shear-stress dependent, supportingthe idea that the transient decrease was related to the convectiveeffect of the step change in flow.

Following the initial decrease, the concentration increased to a newsteady-state that was higher than the pre-stimulus level. The ability tomeasure changes in NO concentration in real-time allows analysis of thekinetics of the responses of endothelial cells to changes in shearstress. The time constants characterizing the rate at which theconcentration approached the steady-state decreased significantly as thesize of the step change in shear stress increased. This suggested thatin addition to the steady-state production rate being dependent on shearstress, the rate at which the signaling process leading to increasedproduction are activated was also dependent on the size of the shearstress stimulus. The simulation of the time course of NO concentrationchanges due to an instantaneous increase in production in response to astep in shear stress indicated that there was negligible transport lagin our measurement system. Therefore, the time dependence of theconcentration changes reflected the dynamics of the cellular response.

For the steady-state concentration to increase with increased shearstress, the stimulated NO production rate must exceed the rate ofremoval by the increased convective transport effects. The experimentaldata and analysis showed that for small increase in shear stress, theconcentration deceased despite an increase in production rate. This wasconsistent with previous study predictions. Furthermore, even though thesteady-state concentration changes for 6 and 10 dyn/cm² were similar,the changes in production rate at 10 dyn/cm² were much greater than at 6dyn/cm², as shown in Table 1 and FIG. 12( b).

The relationships between steady-state NO concentration and productionrate for different flow conditions presented in FIG. 11, provided atemplate for interpreting the measured responses in terms of stimulatedchanges in production. Use of this template required knowledge of theabsolute concentration in the baseline condition. This could bedetermined through the development of an in situ calibration procedure.Alternatively, as accomplished in the present experiment, the basalproduction rate was estimated by fitting a theoretical relationshipbetween production rate and shear stress to the measured NO changes fora range of shear stress step changes. In the study, as shown in Table 1,the highest value for R_(NO) occurred at τ=20 dyn/cm². The technique ofthe present experiment permitted observation of the dynamics of theR_(NO) response on a small time scale. The experimental measurementscombined with mathematical modeling of the transport processes alsoenabled determination of the dynamic changes in NO production by thecells.

Example 2

Direct, real-time measurement of NO concentration changes due toflow-induced shear stress stimulation of endothelial cells in vitrousing the flow chamber of the present invention was performed. In thisstudy, solutions of Dulbecco's Phosphate Buffered Saline (PBS) withcalcium/magnesium (Sigma), supplemented with 70 μM L-arginine (L-arg)(Sigma) or 1 mM L-NAME solution (Sigma) were tested using the flowchamber in the same manner as described in Example 1, with the exceptionthat the NO was measured in response to step changes in flow ratecorresponding to shear stresses from about 1 dyn/cm² to about 5 dyn/cm²and from about 1 dyn/cm² to about 10 dyn/cm², the same experimentalprotocol as that of Example 1 was utilized.

The step change from about 1 to about 5 dyn/cm² produced an increase inNO that is approximately 70% of the response to 10 dyn/cm². Endothelialcells exposed to shear stress in the presence of 1 mM No methyl ester(L-NAME) significantly reduced the flow induce NO response, consistentwith its action as a competitive inhibitor of NO synthase.

It is to be understood that even though numerous characteristics andadvantages of the present invention have been set forth in the foregoingdescription, together with details of the structure and function of theinvention, the disclosure is illustrative only, and changes may be madein detail, especially in matters of shape, size and arrangement of partswithin the principles of the invention to the full extent indicated bythe broad general meaning of the terms in which the appended claims areexpressed.

1. A flow chamber for detecting an analyte comprising: a firstcompartment, wherein said first compartment comprises: a fluid inlet anda fluid outlet for allowing a fluid to flow through said firstcompartment; a second compartment; a first analyte sensor positionedwithin said second compartment for detecting an analyte; a permeablemembrane, wherein said permeable membrane comprises: a first surfacethat is exposed to fluid flow in said first compartment; and a secondsurface in said second compartment, wherein said permeable membraneseparates said first compartment from said second compartment.
 2. Theflow chamber of claim 1, wherein said first analyte sensor is positionedat a known predetermined distance from said first surface of saidmembrane.
 3. The flow chamber of claim 2, wherein said predetermineddistance is about 5 μm to about 50 μm.
 4. The flow chamber of claim 1,wherein a distal end of said first analyte sensor contacts said secondsurface of said membrane.
 5. The flow chamber of claim 1, wherein saidsecond compartment comprises a fluid inlet and a fluid outlet to permitrinsing of said second compartment with a fluid.
 6. The flow chamber ofclaim 1, wherein said first analyte sensor is adapted to detect a smallmolecule selected from the group consisting of: dissolved gases, ions,sugars, nucleotides, proteins and lipids.
 7. The flow chamber of claim1, further comprising a second analyte sensor positioned within saidfirst compartment, wherein said first and second analyte sensors areadapted to detect different analytes.
 8. The flow chamber of claim 1,further comprising a sensor holder associated said second compartmentand said first analyte sensor for positioning said first analyte sensorin said second compartment.
 9. The flow chamber of claim 1, wherein saidsecond compartment is a substantially enclosed internal space having avolume less than about 1 mL.
 10. A flow chamber for detecting an analytecomprising: a first compartment, wherein said first compartmentcomprises: a fluid inlet and fluid outlet for allowing a fluid to flowthrough said first compartment; a second compartment; an analyte sensorpositioned within said second compartment for detecting an analyte; anda structure for allowing passage of an analyte from said firstcompartment to said second compartment, wherein said analyte sensor ispositioned at a known distance from a surface of said structure.
 11. Theflow chamber of claim 10, wherein first analyte sensor is removablyfixed in place by a fastener.
 12. The flow chamber of claim 10, whereinsaid predetermined distance is about 5 μm to about 50 μm.
 13. A methodfor detecting an analyte using a flow chamber comprising: positioning aplurality of cells within the flow chamber of claim 1 on said firstsurface of the permeable membrane; flowing a fluid through said firstcompartment; and detecting, in said first compartment, one more analytesproduced by said plurality of cells.
 14. The method of claim 13, whereinsaid permeable membrane allows for passage of said analyte and whereinsaid analyte is produced by said plurality of cells.
 15. The method ofclaim 13, wherein said analyte is produced by said plurality of cells inresponse to said fluid flow through said second compartment.
 16. Themethod of claim 13, further comprising a step of determining aconcentration of said analyte produced by said plurality of cells. 17.The method of claim 13, further comprising a step of determining achange in concentration of said analyte produced by said plurality ofcells.
 18. The method of claim 13, further comprising a step ofmonitoring an environmental condition of one of said first or secondcompartments using an environmental sensor.
 19. The method of claim 18,further comprising a step of adjusting said environmental condition ofsaid first or second compartments responsive to information obtained insaid monitoring step.
 20. The method of claim 13, further comprising astep of rinsing said second compartment with a fluid betweenmeasurements.